Dihydrofolate reductase is the site of action of several important drugs (i.e., methotrexate) widely used in the chemotherapeutic treatment of numerous malignancies, certain bacterial infections, psoriasis, arthritis and other human diseases. Understanding the detailed mechanism of action of this enzyme and its inhibition by these drugs as well as its unique molecular biology should facilitate a rational approach to drug design and chemotherapy. Thus we continue our studies on the isolation and characterization of hepatic dihydrofolate reductase from various animals, i.e., chicken, beef, pork, sheep, with the rationalization that the comparative investigation of a seemingly unique feature exhibited by an enzyme from a particular animal species might lead to an overall synthesis of understanding of the general nature of this unusual enzyme. Preliminary studies on the purification and properties of sheep liver dihydrofolate reductase suggests that the reductase reaction itself as well as the effects of ionic strength and chaotropic agents (guanidine hydrochloride) are quite different than observed with either the chicken or bovine liver enzymes. The reaction rate is not linear as is the case with the reductase from chicken and beef. Increasing concentrations of KC1 eliminate this lag resulting in an apparent stimulation of the initial rate (2X). Guanidine hydrochloride also linearizes the initial rate however the degree of stimulation (at about 0.7M) is 4 to 5-fold greater than the neutral salt effect. It is postulated that these effects are related to the binding of NADPH and NADP to dihydrofolate reductase.